The long-term objective of this research is to understand the mechanisms responsible for insertion and removal of ribonucleoside monophosphates (rNMPs) from chromosomal DNA. The accurate duplication of genetic material is essential for all living cells. Recently, it has become apparent that DNA polymerases directly incorporate ribose sugars into DNA as rNMPs. A hallmark of DNA is that it is chemically stable and much less reactive than RNA. The 2' hydroxyl on the ribose sugar causes rNMPs to be 100,000 fold more reactive resulting in hydrolysis and DNA breaks under normal physiological conditions. Furthermore, the intracellular concentration of rNTPs far exceeds that of dNTPs contributing to their misinsertion into chromosomal DNA during replication. Error rates for rNMP incorporation suggest misincorporation occurs every ~103 correctly paired bases making rNMP errors far exceed that of any type of replication error or damaged base in vivo. We have found that the replicative DNA polymerases in bacteria frequently incorporation rNMPs into DNA. In this work, we will elucidate the mechanisms of insertion, removal, and the consequences to genome integrity when rNMPs are left unrepaired. Moreover, we have found a novel protein that links rNMP removal to genome integrity providing an evolutionary benefit for rNMP errors. Incorporated rNMPs have profound effects on human health. Ribonucleoside monophosphates slow DNA synthesis and rNMPs have mutagenic potential. Furthermore, RNase H2 the enzyme responsible for removing single rNMPs from DNA is essential in mice and mutations in human RNase H2 results in a neurological disorder known as Aicardi-Goutieres syndrome. Thus, our studies of rNMP insertion and removal have practical implication for human health. Our specific aims are: 1) to determine the rate of rNMP incorporation in vitro; 2) determine the mechanisms of rNMP removal; 3) determine the evolutionary benefit of rNMP removal to genome integrity.